ABSTRACT
Western blotting (WB) and immunohistochemical staining (IHC) are common techniques for determining tissue protein expression. Both techniques require a primary antibody specific for the protein in question. WB data is band(s) on a membrane while IHC result is a staining on a tissue section. Most human genes are known to produce multiple protein isoforms; in agreement with that, multiple bands are often found on the WB membrane. However, a common but unspoken practice in WB is to cut away the extra band(s) and present for publication only the band of interest, which implies to the readers that only one form of protein is expressed and thus the data interpretation is straightforward. Similarly, few IHC studies discuss whether the antibody used is isoform-specific and whether the positive staining is derived from only one isoform. Currently, there is no reliable technique to determine the isoform-specificity of an antibody, especially for IHC. Therefore, cutting away extra band(s) on the membrane usually is a form of misconduct in WB, and a positive staining in IHC only indicates the presence of protein product(s) of the to-be-interrogated gene, and not necessarily the presence of the isoform of interest. We suggest that data of WB and IHC involving only one antibody should not be published and that relevant reports should discuss whether there may be protein multiplicity and whether the antibody used is isoform-specific. Hopefully, techniques will soon emerge that allow determination of not only the presence of protein products of genes but also the isoforms expressed.
Subject(s)
Alternative Splicing , Blotting, Western/ethics , Immunohistochemistry/ethics , Scientific Misconduct , Antibodies/chemistry , Antibody Specificity , Bias , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Mutant Chimeric Proteins/genetics , Mutant Chimeric Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
Este trabajo tuvo como objetivo evaluar la técnica de inmunoelectrotransferencia (Western Blot) para detectar los antígenos específicos de excreción/secreción de Hymenolepis nana en sueros de pacientes con himenolepiosis y con otras helmintiosis confirmadas. Se utilizó a Mesocricetus auratus "hamster" para obtener ejemplares adultos de H. nana. Los antígenos de excreción/secreción fueron obtenidos en el medio MEM (Minimum Essential Medium Eagle), y enfrentados con un grupo de sueros de pacientes con himenolepiosis confirmada para evaluar su calidad inmunológica y con sueros individuales de pacientes con himenolepiosis y con otras helmintiosis confirmadas para detectar mediante la técnica de "Western Blot", los antígenos específicos de este cestode. El grupo de sueros de pacientes con himenolepiosis confirmada reconoció las bandas antigénicas de 50,1; 42,6; 38,9; 32,9; 26,3; 22,4 y 18,6 kDa; sin embargo, los sueros individuales reconocieron diferente número de bandas, siendo la de 50,1 KDa la que fue reconocida por todos ellos. Los sueros de pacientes con helmintiosis confirmadas no reconocieron la banda de 50,1 kDa; sin embargo, dieron reacción cruzada con algunas de las demás bandas, a excepción de los sueros de pacientes con cisticercosis que no reconocieron a ninguna de las bandas de estos antígenos. Se concluye que el antígeno de excreción/secreción de H. nana de 50,1 kDa es específico de este cestode por ser reconocido por todos los sueros de pacientes con himenolepiosis confirmada y no por sueros de pacientes con otras helmintiosis utilizando la técnica de "Western Blot".
Enzyme-linked immunoelectrotransfer blot assay or "Western blot" was evaluated to detect specific excretory/secretory antigens from Hymenolepis nana using sera from patient with hymenolepiosis and other confirmed helminthiosis. Hymenolepis nana adults were obtained from experimentally infected golden hamster, the parasits were cultured in Minimum Essential Medium Eagle to get excretory/secretory antigens which were faced to a pool of sera from patients with confirmed hymenolepiosis to evaluate its immunological quality and with individual sera from patients with hymenolepiosis and other confirmed helminthiosis to detect by mean "Western blot" technique, this parasite specific antigens. The pool of sera from patients with confirmed hymenolepiosis recognized seven antigenic bands; 50,1; 42,6; 38.9; 26,3; 22,4; and 18,6 kDa; however, the individual sera recognized different number of bands being 50,1 kDa band the most recognized by all of them. The sera from patients with confirmed helminthiosis did not recognized 50,1 KDa band; however they gave cross reaction with some of the other bands, except the sera from patients with cysticercosis which did not recognized any of this antigen bands. Consequently 50,1 kDa antigen is specific because is recognized by all the sera from patients with confirmed hymenolepiosis and is not recognized by sera from patients with other helminthiosis using "Western blot" technique.
